JP2019531264A - Composition for promoting melanin containing specific siRNA - Google Patents

Abstract

In one aspect, the present invention relates to a composition for blackening hair, preventing whitening of hair, preventing or treating melanin-lowering diseases, which contains siRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. According to one aspect of the present invention, the hair-related industry can be developed and the welfare of related consumers can be improved. [Selection] Figure 1

Description

  The present specification relates to a composition for blackening hair, preventing melanin-lowering disease, or improving melanin-lowering disease, which contains a specific siRNA.

  Melanin is a phenolic biopolymeric substance with a complex form of black pigment and protein, browning that occurs when the cut surfaces of apples, potatoes, and bananas are exposed to the air or Observed in animal coat hair, skin, hair, eyes, etc. When melanin is excessively produced and deposited on the skin, it can cause stains, freckles, etc., and is directly connected to hair darkening, and melanin also promotes skin or hair aging and may induce skin cancer.

  Melanocyte stimulating hormone (MSH) is secreted by ultraviolet rays, inflammation, hormones, etc., and the MSH reacts with a receptor to improve cAMP in melanocytes and synthesize melanin. Is secreted to the outside of melanocytes and plays a role of protecting the skin or hair from ultraviolet rays and the like. Melanin synthesis is mainly regulated by α-MSH, and MITF, TYR, TRP1, TRP2, and the like are known as proteins involved in melanin synthesis.

  Melanin in the hair is an important factor that determines the hue of the hair. The melanin produced in the melanocytes in the hair follicle is transferred to the keratinocytes of the hair cortical cells and then moves upward with the growth of the hair. To do. Hair whitening, recognized as a phenomenon of physiological aging, is induced by a decrease in melanogenesis, such as by a numerical decrease in hair melanocytes and a decrease in function of melanocytes. Currently, hair dyeing is used as a solution for whitening hair, but it is a temporary method that requires re-dying as the hair grows, and is included in the hair dye. Ingredients become stimulating ingredients, causing contact dermatitis and skin allergies.

  In addition, when abnormality occurs in melanin biosynthesis, abnormal whitening of the skin appears due to abnormal coloring, thereby causing diseases such as whitening (albinism) and vitiligo.

  On the other hand, siRNA (small interfering RNA), which is an interfering RNA composed of about 10 to 60 nucleic acids, binds to RISC (RNA-induced silencing complex) or the like in the cell, and complementary base sequence in the cell. It is well known that it has an RNA interference action that specifically acts on mRNA having a function to suppress target mRNA and the like. siRNA can inhibit gene expression even in an amount about 4 to 10 times less than antisense oligonucleotide, has low cytotoxicity and excellent selective inhibition of gene, and can be used for cancer and genetic diseases. Inhibiting the expression of a protein by a gene that induces the disease can eliminate the cause of the disease, and thus attracts much interest as a new effective disease therapeutic agent.

Korean Published Patent No. 10-2016-0066986 Korean Published Patent No. 10-2011-0130282

ALLOUCHE, J. et al., “In Vitro Modeling of Hyperpigmentation Associated to Neurofibromatosis Type 1 Using Melanocytes Derived from Human Embryonic Stem Cells”, Preceedings of the National Academy of Sciences, 2015, vol. 112, no. 29, pages 9034- 9039 ROY, R. et al., “Association between Risk of Oral Precancer and Genetic Variations in MicroRNA and Related Processing Genes”, Journal of Biomedical Science, 2014, vol. 21, thesis no. 48, internal pages 1-7 LI, Y. et al., “VIT1 / FBOX11 Knockdown Induces Morphological Alterations and Apoptosis in B10BR Mouse Melanocytes”, International Journal of Melecular Medicine, 2009, Vol. 23, pages 673-678

  In one aspect, the present invention provides hair blackening, hair whitening prevention, melanin-lowering disease prevention, melanin-lowering disease containing siRNA comprising the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient The purpose of the present invention is to provide a composition for the treatment of melanin or to improve melanin lowering disease, to develop the white hair care field and the melanin lowering disease related field, and to promote the welfare of related consumers and patients To do.

  In order to solve the above-mentioned problems, the present invention provides, in one aspect, for hair blackening, hair whitening prevention, melanin-lowering disease containing siRNA comprising the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. Provided is a composition for prevention, treatment of melanin-lowering disease, or improvement of melanin-lowering disease.

  In another aspect, the present invention provides a method for blackening hair, a method for preventing whitening of hair, a melanin-lowering disease, comprising administering to a subject a composition comprising siRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The prevention method of this, the treatment method of a melanin lowering disease, or the improvement method of a melanin lowering disease is provided.

  In another aspect, the present invention is effective for use in blackening hair, preventing whitening of hair, preventing melanin-lowering disease, treating melanin-lowering disease, or improving melanin-lowering disease. Provided are siRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as a component and a composition comprising the same.

  In another aspect, the present invention provides SEQ ID NO: 1 as an active ingredient for hair blackening, hair whitening prevention, prevention of melanin lowering disease, treatment of melanin lowering disease, or improvement of melanin lowering disease. Alternatively, the present invention provides a non-therapeutic cosmetic use of siRNA consisting of the nucleotide sequence of SEQ ID NO: 2 and a composition comprising the same. In another aspect, the present invention provides a composition for blackening hair, a composition for preventing whitening of hair, a composition for preventing melanin-lowering disease, a composition for treating melanin-lowering disease, or a melanin-lowering disease. The use of siRNA which consists of a base sequence of sequence number 1 or sequence number 2 for using for manufacture of the composition for improvement of this is provided.

  In another aspect of the present invention, the composition may be a pharmaceutical composition or a cosmetic composition.

  When using the composition containing siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient according to one aspect of the present invention, hair blackening, hair whitening prevention, prevention of melanin-lowering diseases, or Treatment and improvement of melanin lowering disease can be effectively achieved.

It is a figure which shows that the content of melanin increases notably by siRNA which consists of a base sequence of sequence number 1 or 2 in a melan-a cell. This is a Western blot document showing that tyrosinase and TRP1 are markedly increased by siRNA having the nucleotide sequence of SEQ ID NO: 1 or 2 in Melan-a cells. It is the figure which showed that the content of melanin increases markedly by siRNA which consists of a base sequence of sequence number 1 or 2 in B16F1 cell. This is a Western blot document showing that tyrosinase and TRP1 are remarkably increased by siRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 in B16F1 cells.

  Hereinafter, this specification will be described in detail.

  As used herein, “hair” refers to a thread-like structure composed of keratinized epithelial cells and coming out on the surface of the skin, and specifically refers to body hair, including hair and body hair. This is a broad concept.

  In the present specification, “skin” means a tissue covering the body surface of an animal, and is the broadest concept including not only the tissue covering the body surface such as the face or body but also the scalp and hair.

  In this specification, “blackening” means that the colored pigment of skin or hair increases, and in particular, the phenomenon that the amount of melanin increases and the skin or hair becomes darker. Can mean. For example, the blackening of hair includes a phenomenon in which the hair turns black, and includes prevention and prevention of whitening of the hair and white hair.

  In the present specification, “whitening” means that the colored pigments of the skin or hair are reduced, and in particular, the phenomenon that the amount of melanin pigments is reduced and the skin or hair becomes lighter in color. Can mean. For example, whitening or whitening of hair includes a phenomenon that the hair turns white.

  In the present specification, “hair darkening effect substance” or “hair whitening prevention effect substance” is a generic term for substances that darken hair brightness and prevent further whitening of hair. It may be. In addition, it may be a generic term for substances that act to induce, improve and enhance the deposition of colored pigments, particularly melanin, and may be a single compound, polymer, DNA, RNA, siRNA, miRNA, peptide, Although it may contain protein etc., it is not restricted to these.

  In the present specification, “melanin-lowering disease” refers to a disease in which melanin deposition is abnormally suppressed due to abnormal synthesis or degradation of melanin pigment, melanin production is reduced, or melanin is excessively removed. Including. For example, whitening (Albinism), leukoderma, partial albinism, tinea, vitiligo, quadrichrome vitiligo, vitiligo pontue, idiopathic drops Hypomelanosis, punctate albinism, symptomatic albinism (e.g., Alezandrinisindrome, Hermansky-Pudlack syndrome, Chediak group) (Chediak-Higashi syndrome), Griscelli syndrome (Elegende syndrome), Riselli syndrome type 2 (Griscelli syndrome type 2) and Griscelli syndrome type 3 (Wardenburg syndrome), Tightzre-skin group Group (Cross-MuKusick-Brain syndrome), ABCD syndrome group, Albinism-deafness syndrome, and Vogt-Koyanadomi group, Vogt-Koyaradigi group Oculocutaneous albinism, hypomelanosis (hy omelanosis (idiopathic gutate hypomelanosis), phylloid hypomelanosis, progressive macula hypoderma, melanosis Nevus depimentosus, post-inflammation hypopigmentation (post-inflammation hyperpigmentation), whiteness eruption (Vagabond's leuomen) Dementia syndrome (Ymenite deaf) -Blind hypopigmentation syndrome, Wende-Buckus syndrome, Woronoff's ring, melanin deficiency (amelanism), and leucism.

  In the present specification, the “effective substance for preventing, improving and treating melanin lowering disease” is a substance which prevents or suppresses the morbidity of melanin lowering disease in advance, a substance which reduces or alleviates the symptoms of the affected melanin lowering disease, Contains substances that eliminate the causes of melanin-lowering diseases, substances that promote melanin synthesis, substances that prevent the suppression of melanin synthesis, substances that suppress the degradation of melanin, and include the entire skin, entire hair, and specific hair Or a generic term for substances that act to darken the brightness of specific skin sites, induce, improve and enhance the deposition of melanin pigments, single compounds, polymers, DNA, RNA, It may include, but is not limited to, siRNA, miRNA, peptide, protein and the like.

  In one aspect, the present invention provides hair blackening, hair whitening prevention, melanin-lowering disease prevention, melanin-lowering disease containing siRNA comprising the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient The present invention provides a composition for the treatment of or a melanin lowering disease.

According to an exemplary embodiment, the siRNA including the base sequence of SEQ ID NO: 1 may be represented by “# 1”, and the siRNA including the base sequence of SEQ ID NO: 2 may be represented by “# 2”. The sequence of siRNA may include the following base sequence.
Base sequence of # 1 (SEQ ID NO: 1): 5′-CCAACCCAGAACAAGCAGAACA-3 ′
Base sequence of # 2 (SEQ ID NO: 2): 5′-GAACACGAAGGCUGUAACAAAA-3 ′

  The siRNA may include a base sequence having 80%, preferably 90%, and more preferably 100% homology with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

  The siRNA may be an independent single-stranded RNA containing the sequence or a sequence having 80 to 100% homology with the sequence, or may be a double-stranded RNA. The siRNA includes a sense strand RNA and an antisense strand RNA complementary thereto, or a single strand RNA having a stem-loop structure in which the sense strand RNA and the antisense strand RNA are connected by a loop. There may be. The double strands or stem sites may contain unpaired parts due to mismatches (corresponding bases are not complementary), bulges (no bases corresponding to one strand), or the like. The total length of the siRNA is 10 to 60 bases, preferably 15 to 40 bases, and more preferably 18 to 30 bases. Further, the loop region has no special meaning in the sequence, and it is sufficient to have about 3 to 10 bases in order to simply link the sequences at appropriate intervals. The structure of the siRNA end can be either a blunt end or a cohesive end. As the structure of the sticky end, either a structure in which the 3 'end protrudes (protruding structure) and / or a structure in which the 5' end protrudes is possible, and the number of protruding bases is not limited. In addition, siRNA can be used as long as the expression suppression effect of the target gene can be maintained. ) May be included. The siRNA terminal structure does not need to have a cleavage structure on both sides, and may be a structure in which one terminal site of siRNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with the pair of stem portions.

  Furthermore, in the siRNA according to one aspect of the present invention, the RNA strand can include at least one chemical modification within its sugar moiety, nucleobase moiety or internucleotide structure. Such deformation can make it possible to inhibit the destruction of siRNA by nucleases in vivo. Any chemical modification capable of improving the stability and biocompatibility of siRNA according to one aspect of the present invention in vivo is included in the scope of the present invention. Among the preferred variations on the sugar moiety, mention may be made of the 2 ′ position of ribose such as 2′-deoxy, 2′-fluoro, 2′-amino, 2′-thio, or 2′-O-alkyl. And preferably a variation that occurs in the presence of a 2′-O-methyl or LNA that replaces the normal 2′-OH group on the ribonucleotide and a methylene bridge between the 2 ′ and 4 ′ positions of the LNA. In the case of nucleobase, 5-bromo-uridine, 5-iodo-uridine, N3-methyl-uridine, 2,6-diaminopurine (DAP, 5-methyl-2′-deoxycytidine, 5- (1-propynyl)- A modified base such as 2'-deoxy-uridine (pdU), 5- (1-propynyl) -2'-deoxycytidine (pdC) or a base conjugated with cholesterol can be used. Preferred variants of include phosphorothioate, methylphosphonate, those that replace the phosphodiester group in the backbone by a phosphorodiamidate group, or N- (2-aminoethyl) -glycine linked by a peptide bond ( PNA, peptide nucleic acid) Variously modified bases, sugars, backbones are modified morpholino type modified nucleic acids (bases fixed to the morpholine ring and linked by phosphorodiamidate groups) or PNA (peptide bonds To a N- (2-aminoethyl) -glycine unit linked to the base).

  The siRNA according to the present invention includes “isolated” and “coupled” to other transmission and transport means and the like, which means that it is not in a natural state, It means that it may be obtained by any means related to human intervention, including what is required. In particular, the siRNA according to the present invention can be produced using chemical synthesis, enzymatic methods, polymerase chain reaction (PCR), and can be produced by the amplification of specific nucleotide sequences or various existing sequences by recombinant synthesis. It may be obtained by purifying a nucleic acid structure or siRNA.

According to another embodiment, the siRNA may enhance melanin production or deposition.

  According to another embodiment, the siRNA may enhance the expression of one or more of tyrosinase and TRP1 (Tyrosinase-Related Protein 1).

  The siRNA according to one aspect of the present invention is a TRP1 protein that promotes melanin production or deposition in melanocytes that are melanocytes and B16F1 cells, and that is involved in the formation of melanin and an enzyme that produces melanin. It was confirmed that it promotes the expression of. In addition, when the siRNA is treated, the number of cells expressing tyrosinase or TRP1 may be increased. Tyrosinase is an enzyme involved in the formation of melanin and the like, and when tyrosinase expression is enhanced, the production or deposition of melanin increases. TRP1 is a protein involved in the synthesis of melanin, and like tyrosinase, when the expression of TRP1 is increased, the production or deposition of melanin increases. Melanin in hair is an important factor that determines the hue of hair, and melanin produced in melanocytes in hair follicles is transferred to keratinocytes in hair cortical cells and then moves upward with hair growth Therefore, hair whitening is induced by a decrease in the production of melanin, such as by a numerical decrease in hair melanocytes and a decrease in function of melanocytes. Through the siRNA, the production of melanin can be promoted and the expression of enzymes and proteins involved in the melanin can be promoted, so that the skin and hair can be blackened, and the skin and hair can be prevented from whitening. can do.

  In addition, the present invention, in one aspect, can promote the production of melanin and increase the expression of enzymes and proteins involved in the siRNA via the siRNA, thus preventing melanin-lowering diseases and symptoms, It has also been clarified that it can be treated and improved.

  According to another aspect of the present invention, the composition may further comprise any carrier that allows for improved biocompatibility and transmissibility of the siRNA according to the present invention. In particular, carriers that can be used with siRNA include liposomes, exosomes, peptides (eg, cell-permeable peptides) and various nanoparticles. In addition, a recombinant vector, a plasmid, or the like containing the siRNA can also be used as a transmission or transport means. Various techniques and methods known in the art can be applied to the transmission or carrying means, and deformation, improvement, etc. can be applied depending on the dosage form.

  The siRNA is preferably operably linked to at least a promoter for proper transcription in a transferred cell, but is not limited thereto. The promoter is not particularly limited as long as it can function in cells. The siRNA according to one aspect of the present invention includes a leader sequence, a polyadenylation sequence, a promoter, an enhancer, an upstream activation sequence, a signal peptide sequence, and a transcription terminator as necessary for efficient action. It may further contain regulatory sequences.

  According to another aspect of the present invention, the content of siRNA in the composition may be in the range of 0.000000001% by mass to 20% by mass with respect to the total mass of the composition. The content is 0.000000001 mass% or more, 0.000000005 mass% or more, 0.00000001 mass% or more, 0.00000005 mass% or more, 0.0000001 mass% or more, based on the total mass of the composition. 0.0000005% by mass or more, 0.000001% by mass or more, 0.000005% by mass or more, 0.00001% by mass or more, 0.00005% by mass or more, 0.0001% by mass or more, 0.0005% by mass or more, 0 0.001% or more, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.3% or more, 0.5% or more, 8% by weight or more, 1% by weight or more, 3% by weight or more, 5% by weight or more, 8% by weight or more, 10% by weight or more, 12% by weight or more, 15% by weight or more, or 18% by weight It may be the top. The content is 20% by mass or less, 18% by mass or less, 15% by mass or less, 12% by mass or less, 10% by mass or less, 8% by mass or less, 5% by mass or less, based on the total mass of the composition. 3 mass% or less, 1 mass% or less, 0.8 mass% or less, 0.5 mass% or less, 0.3 mass% or less, 0.1 mass% or less, 0.05 mass% or less, 0.01 mass% 0.005 mass% or less, 0.001 mass% or less, 0.0005 mass% or less, 0.0001 mass% or less, 0.00005 mass% or less, 0.00001 mass% or less, 0.000005 mass% or less 0.000001 mass% or less, 0.0000005 mass% or less, 0.0000001 mass% or less, 0.00000005 mass% or less, 0.00000001 mass% or less, or 0.000000005 mass% or less.

  According to still another aspect of the present invention, the dosage of the siRNA may range from 0.000001 mg / kg / day to 20 mg / kg / day. The dose is 0.000001 mg / kg / day or more, 0.000005 mg / kg / day or more, 0.00001 mg / kg / day or more, 0.00005 mg / kg / day or more, 0.00001 mg / kg / day. Or more, 0.0005 mg / kg / day or more, 0.0001 mg / kg / day or more, 0.0005 mg / kg / day or more, 0.001 mg / kg / day or more, 0.005 mg / kg / day or more, 0.01 mg / Kg / day or more, 0.05 mg / kg / day or more, 0.1 mg / kg / day or more, 0.5 mg / kg / day or more, 0.8 mg / kg / day or more, 1 mg / kg / day or more, 2 mg / Kg / day or more, 3 mg / kg / day or more, 5 mg / kg / day or more, 8 mg / kg / day or more, 10 mg / kg / day or more, 12 mg / kg / day or more, 15 mg / kg / day or more, It may be at 18mg / kg / day or more. The dose is 20 mg / kg / day or less, 18 mg / kg / day or less, 15 mg / kg / day or less, 12 mg / kg / day or less, 10 mg / kg / day or less, 8 mg / kg / day or less, 5 mg / Kg / day or less, 3 mg / kg / day or less, 2 mg / kg / day or less, 1 mg / kg / day or less, 0.8 mg / kg / day or less, 0.5 mg / kg / day or less, 0.1 mg / kg / Day or less, 0.05 mg / kg / day or less, 0.01 mg / kg / day or less, 0.005 mg / kg / day or less, 0.001 mg / kg / day or less, 0.0005 mg / kg / day or less, 0 It may be 0.0001 mg / kg / day or less, 0.00005 mg / kg / day or less, 0.00001 mg / kg / day or less, or 0.000005 mg / kg / day or less. Determination of the dose of the active ingredient is within the level of ordinary technicians, and the daily dose of the drug varies depending on the degree of progression of the subject to be administered, the time of onset, age, health condition, complications, etc. Depending on different factors.

  In another aspect, the present invention may be a composition for the prevention, treatment and amelioration of a melanin-lowering disease comprising, as an active ingredient, siRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

  According to one embodiment, the melanin-lowering disease is vitiligo, depigmented nevus, white urticaria, white shark, post-inflammation depigmentation, maculopathy, partial leukoderma, idiopathic icy low It may be one or more selected from the group consisting of melanosis and punctate albinism. Melanin is observed in the outer coat hair, hair, skin, head, eyes, etc. of animals. When melanin production is insufficient, deposition is suppressed, or excessive degradation occurs, melanin-lowering disease develops. There are things to do. In one aspect, the present invention enhances the production or deposition of melanin and enhances the expression of tyrosinase and TRP1, and thus can be used for the prevention, improvement, and treatment of the melanin lowering disease.

  According to one aspect of the invention, the composition may be a pharmaceutical composition.

  The pharmaceutical composition comprises a pharmaceutical adjuvant such as a carrier, excipient, preservative, preservative, stabilizer, wettable powder or emulsifier, salt and / or buffer for adjusting osmotic pressure, In addition, it may further contain other therapeutically useful substances, and may be formulated in various oral or parenteral dosage forms by conventional methods.

  Examples of the oral administration agent include tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, pellets, and the like. Forms include active ingredients, surfactants, diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts) , As well as polyethylene glycol). The tablets may further contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally disintegrating agents such as starch, agar, alginic acid or its sodium salt. , And may contain pharmaceutical additives such as absorbents, colorants, flavoring agents, and sweetening agents. The tablets may be manufactured by conventional mixing, granulating or coating methods.

  The parenteral administration form may be a transdermal dosage form, for example, an injection, infusion, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, patch. The dosage form may be, but is not limited to these.

  The pharmaceutical composition may be administered orally or parenterally, and when parenterally administered, select an external skin injection, intraperitoneal, rectal, vein, muscle, subcutaneous, intradermal, intrathoracic or intracerebral intravascular injection method You can do it.

  The pharmaceutical composition may be an external preparation for skin, and the external preparation for skin is a general term for compositions that can be applied to the outer skin, and is a generic term for pharmaceuticals or external pharmaceutical products in various dosage forms. Can be included in this.

  According to another aspect of the present invention, the composition may be a cosmetic composition for blackening hair, preventing whitening of hair, preventing or improving melanin-lowering diseases.

  The cosmetic composition may further include functional additives and components included in general cosmetic compositions. The functional additive may contain a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extracts. Other ingredients included are oil and fat ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjustment. Agents, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like.

  The dosage form of the cosmetic composition is not particularly limited, and may be appropriately selected according to the intended purpose. For example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing foam, It may be produced in any one or more dosage forms selected from the group consisting of cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto.

  When the dosage form according to one aspect of the present invention is a paste, cream, or gel, the carrier component is animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, Talc or zinc oxide may be used.

  When the dosage form according to one aspect of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, particularly when it is a spray. May further contain a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

  When the dosage form according to one aspect of the present invention is a solution or an emulsion, a solvent, a solvating agent, or an emulsifying agent is used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, There are ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycols, or fatty acid esters of sorbitan.

  When the dosage form according to one aspect of the present invention is a suspension, the carrier component includes a liquid diluent such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and poly Suspending agents such as oxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth may be used.

  When the dosage form according to one aspect of the present invention is a surfactant-containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinate monoester, isethionic acid, imidazolium derivative, Methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used.

  According to another aspect of the invention, the composition may be a food or health food composition.

  The food or health food composition may be in a liquid or solid dosage form, for example, various foods, beverages, gum, tea, vitamin complex, health functional foods, health supplements, etc. , Powders, granules, tablets, capsules or beverages. The food composition of each dosage form may contain ingredients that are usually used in the field in addition to the active ingredients as appropriate by the person skilled in the art depending on the dosage form or purpose of use. When applied at the same time as the raw material, there may be a synergistic effect.

  The liquid component that can be contained in addition to the active ingredient disclosed in the present specification is not particularly limited, and may contain various flavoring agents or natural carbohydrates as additional ingredients in the same manner as a normal beverage. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the natural carbohydrate may generally be about 1-20 g per 100 ml of the composition disclosed herein, and in one aspect about 5-12 g.

  In one aspect, the food composition comprises various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its Salts, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be included. In other aspects, it may include pulp for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination. The proportion of the additive may vary, but is generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.

  Hereinafter, the configuration and effect of one aspect of the present invention will be described more specifically through examples and experimental examples. It should be noted that the following examples are merely illustrative for the purpose of helping understanding of the present invention, and the scope and scope of the present invention are not limited thereby.

[Example 1]
Preparation of cells and skin model Normal human epidermal melanocytes, normal human epithelial melanocytes (NHEMs, Cascade Biologicals, Portland, OR, USA) were obtained, and melanocytes from C57BL / 6 J (black, a / a) mice The Melan-A cell line was obtained from Dr. Dorothy C. Bennett (St. George's Hospital Medical School, London, UK). The B16F1 mouse melanoma cell line was obtained from ATCC (Manassas, VA, USA).

  Normal human epidermal melanocytes were maintained in M-254 medium under human melanocyte growth supplements (HMGS) (Cascade Biologics, Inc., Mansfield, UK). Melan-A cells were treated with RPMI 1640 medium in 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin, and 0.2 μM phorbol 12-myristate 13-acetate (phorbol 12-myristate 13- acetate). The B16F1 mouse melanoma cell line was cultured in DMEM supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin.

Reagents and siRNA transfection
α-MSH, arbutin, and kojic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). siRNAs # 1 (5′-CCAACCAGAACAAGCAGAACA-3 ′), # 2 (5′-GAACACGAAGGCUGUAACAAAA-3 ′), and negative control group siRNA (scrambled control siRNA, siNC) (5′-CCUACCGCCACCAAUU) Obtained from Resolution (Seoul, South Korea). SiRNA was transfected into Melan-a and B16F1 cells cultured in 60 mm dishes using Lipofectamine® RNAi MAX (Invitrogen, Carlsbad, CA, USA). After culturing with siRNA for 24 hours, the cells were treated with each compound (reagent) and maintained for 48 hours.

Melanin assay cells were treated with trypsin / EDTA, centrifuged at 1000 × g for 5 minutes, and washed twice with PBS. The final cell pellet in the tube was photographed and the cell pellet was dissolved in 1N NaOH. The homogenized cell extract was transferred to a 96-well plate, and the relative amount of melanin at an absorbance of 405 nm was measured using an ELISA plate reader.

Western Blot Analysis All lysates were prepared using 2 x Laemmli sample buffer (2 x Laemmli sample buffer, 62.5 mM Tris-HCl, pH 6.8, 25% [v / v] glycerol, 2% [w / v] SDS, 5 % [V / v] β-mercaptoethanol and 0.01% [w / v] bromophenol blue) (Bio-Rad, Hercules, CA, USA). All cellular proteins were measured using Bradford solution (Bio-Rad). Thereafter, the samples were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad). After blocking with 4% (w / v) skim milk in TBST (25 mM Tris, 140 mM NaCl, and 0.05% [v / v] Tween® 20), membranes were identified as specific 1 Overnight culture was performed with the next antibody.

  Anti-actin antibody (anti-actin antibody, MAB1501, 1: 10,000) was obtained from Millipore (Temecula, CA, USA) and anti-αPEP7 (tyrosinase) antibody (anti-αPEP7 (tyrosinase) antibody, 1: 1000). And anti-αPEP1 (TRP1) antibody (anti-αPEP1 (TRP1) antibody, 1: 1000) were obtained from V. J. Hearing (NIH, Bethesda, MD). For protein detection, the membrane is cultured using an HRP-conjugated secondary antibody, and the signal is obtained using SuperSignal® West Dura HRP Detection Kit (Pierce, Rockford, IL, USA). Measured.

Statistical analysis Each data was obtained by performing at least three independent experiments and expressed as mean ± SE (standard error). Statistical evaluation of the results was performed using a one-way analysis of variance (1-way ANOVA) (*: p <0.05, **: p <0.01).

[Experimental Example 1] Melan Production by siRNA, Regulation of Related Enzymes and Proteins Melan-a cells were transferred to siRNA (# 1, # 2) and scrambled control siRNA (scrambled control siRNA, siNC), respectively. After 48 hours, the content of melanin was measured after treatment with a specific substance D (0.5 μM) that suppresses the production of melanin or culture under serum-free serum conditions for 2 days. As can be seen from FIG. 1, treatment with a specific substance D that inhibits the production of melanin or melanin-a cell culture under serum-free conditions suppressed the production of melanin in the control group (siNC), but the stimulation (specific The production of melanin suppressed by substance D or serum starvation was significantly enhanced by siRNA (# 1, # 2) (FIG. 1).

  Experiments were also conducted on the expression of melaninase. As a result of Western blot analysis, when melan-a cell siRNA (# 1, # 2) was transfected, the levels of tyrosinase and TRP1 protein were markedly increased compared to wild type cells (FIG. 2).

  B16F1 cells are also transfected with siNC and siRNA (# 1, # 2), and one day later, treated with a specific substance D (0.5 μM) that suppresses melanogenesis or treated with serum-free medium. After a day, the degree of melanin production was confirmed, and tyrosinase and TRP1 were analyzed by Western blot.

  As a result, the production of melanin suppressed by specific substance D or serum starvation in B16F1 cells was remarkably increased by siRNA (# 1, # 2) transfection, similar to melan-a cells (FIG. 3). In addition, expression of tyrosinase and TRP1 suppressed by specific substance D or serum starvation was significantly increased by siRNA (# 1, # 2) transfection (FIG. 4).

  Summing up such results, the siRNA (# 1, # 2) can enhance the synthesis of melanin, and can enhance the expression of melanin-producing enzyme and melanin-producing protein. Since the hair can be blackened and the whitening of the hair can be prevented, the present invention can be used for hair blackening and hair care in one aspect. Moreover, since the production | generation of melanin can be accelerated | stimulated with respect to the specific site | part of the skin or the whole skin using such a characteristic concerning one side of the present invention, a melanin-lowering disease using one side of the present invention Can prevent, treat, and improve symptoms and symptoms.

  Hereinafter, examples of the dosage form of the composition according to one aspect of the present invention will be described, but application to various other dosage forms is possible, and these are not intended to limit the present invention. This is for concrete explanation.

[Form example 1] Soft capsule 10 mg of liposome containing 2 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, L-carnitine 80-140 mg, soybean oil 180 mg, palm oil 2 mg, vegetable hardened oil 8 mg, 4 mg of yellow wax and 6 mg of lecithin were mixed and filled into one capsule according to a usual method to produce a soft capsule.

[Formulation Example 2] Tablet After mixing 10 mg of liposome containing 2 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, galactooligosaccharide 200 mg, lactose 60 mg and maltose 140 mg and granulating using a fluid bed dryer Then, 6 mg of sugar ester was added and tableted by a tableting machine to produce tablets.

[Dosage Form Example 3] Granule 10 mg of liposome containing 2 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 250 mg of anhydrous crystalline glucose and 550 mg of starch are mixed and formed into granules using a fluid bed granulator. After that, it was filled in a package to produce a granule.

[Formulation Example 4] Drink agent After mixing 10 mg of liposome containing 2 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, glucose 10 g, citric acid 0.6 g, and liquid oligosaccharide 25 g, 300 ml of purified water was added. In addition, each bottle is filled with 200 ml. After filling the bottle, it was sterilized at 130 ° C. for 4 to 5 seconds to produce a drink.

[Formulation Example 5] Injectable Injectable according to a usual method using 20 mg of liposomes containing 5 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, an appropriate amount of sterile distilled water for injection, and an appropriate amount of pH regulator. Manufactured.

[Form 6] Soft lotion (skin lotion)
10% by mass of liposomes containing 1.0% by mass of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 1.00% by mass of L-ascorbic acid-2-phosphate magnesium salt, water-soluble collagen (1% aqueous solution 1.00% by mass, sodium citrate 0.10% by mass, citric acid 0.05% by mass, licorice extract 0.20% by mass, 1,3-butylene glycol 3.00% by mass, and purified water as the remaining amount A soft lotion (skin lotion) was produced.

[Formulation Example 7] Cream 10% by mass of liposomes containing 1.00% by mass of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 2.00% by mass of polyethylene glycol monostearate, self-emulsifying monostearic acid Glycerin 5.00% by mass, propylene glycol 4.00% by mass, squalene 6.00% by mass, tri-2-ethylhexaneglyceryl 6.00% by mass, glycosphingolipid 1.00% by mass, 1,3-butylene glycol 7 A cream-form preparation was produced using 0.000% by mass, 5.00% by mass of beeswax, and purified water as the remaining amount.

[Dosage Form Example 8] Pack 10% by mass of liposomes containing 1.00% by mass of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 13.00% by mass of polyvinyl alcohol, L-ascorbic acid-2-phosphate Magnesium salt 1.00% by mass, lauroyl hydroxyproline 1.00% by mass, water-soluble collagen (1% aqueous solution) 2.00% by mass, 1,3-butylene glycol 3.00% by mass, ethanol 5.00% by mass, A pack was produced using purified water as the remaining amount.

[Formulation Example 9] Health Food A health food was produced according to the usual method with the composition described in the following table.

  The composition ratio of the vitamin and inorganic mixture is a mixture composition taking as an example a component that is relatively suitable for health foods, but the blending ratio may be arbitrarily changed, and according to a normal method for producing health foods After mixing the above components, they may be used in the production of a health food composition according to a conventional method.

  [Formulation Example 10] Health drink

  As shown in Table 2, the remaining amount of purified water was added to a total volume of 900 ml, and the ingredients were mixed according to a normal health drink manufacturing method, followed by stirring and heating at 85 ° C. for about 1 hour. The filtrate obtained by filtering the solution was put into a sterilized 2 liter container, sealed and sterilized, and stored refrigerated to produce a health drink.

  Although specific portions of the present invention have been described in detail, such specific descriptions are merely preferred embodiments for those having ordinary skill in the art, and thus the scope of the present invention. It will be clear that is not limited. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

  A composition for blackening hair or preventing whitening of hair, comprising an siRNA comprising the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.   A composition for the prevention and treatment of a melanin-lowering disease comprising, as an active ingredient, siRNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.   The composition according to claim 1 or 2, wherein the siRNA enhances melanin production or deposition.   The composition according to claim 1 or 2, wherein the siRNA enhances expression of one or more of tyrosinase and TRP1.   The composition according to claim 1 or 2, wherein the content of siRNA in the composition is in the range of 0.000000001 mass% to 20 mass% with respect to the total mass of the composition.   The composition according to claim 1 or 2, wherein the dose of the siRNA is in the range of 0.000001 mg / kg / day to 20 mg / kg / day.   The melanin-lowering diseases include: whitening, vitiligo, depigmented nevus, white urticaria, white shark, post-inflammation depigmentation, maculopathy, partial albinism, idiopathic droplet hypomelanosis and The composition according to claim 2, wherein the composition is one or more selected from the group consisting of punctate albinism.   The composition according to claim 1 or 2, wherein the composition is a pharmaceutical composition.   The composition according to claim 1 or 2, wherein the composition is a cosmetic composition for blackening hair, preventing whitening of hair, preventing or improving melanin-lowering diseases.